Nephrin gene and protein

ABSTRACT

The present invention provides for compositions and methods for detecting susceptibility for basement membrane disease, in particular congenital nephrotic syndromes of the Finnish type. The present invention provides for nucleic acids and protein for use in methods and compositions for the diagnosis of disease and identification of small molecule therapeutics for treatment of such disease, in particular of proteinuria associated with kidney disease.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 09/759,622, filed Jan. 12, 2001, now U.S. Pat. No. 7,105,291, which is a divisional of U.S. application Ser. No. 09/040,774, filed Mar. 18, 1998 now U.S. Pat. No. 6,207,811, issued Mar. 21, 2001. The entire teachings of the above applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Congenital nephrotic syndrome of the Finnish type (CNF, NPHS1, MIM 256300) is an autosomal recessive disorder, and a distinct entity among congenital nephrotic syndromes. It is characterized by massive proteinuria at the fetal stage and nephrosis at birth. Importantly, NPHS1 appears to solely affect the kidney and, therefore, it provides a unique model for studies on the glomerular filtration barrier.

The primary barrier for ultrafiltration of plasma in renal glomeruli comprises three layers; a fenestrated endothelium, a 300-350 nm thick glomerular basement membrane (GBM), and slit pores, i.e. diaphragms located between the foot processes of the epithelial cells. This barrier is a highly sophisticated size-selective molecular sieve whose molecular mechanisms of function are still largely unclarified. It is anticipated that the GBM, a tightly cross-linked meshwork of type IV collagen, laminin, nidogen and proteoglycans, contains pores that restrict the penetration of large proteins and cells, and, additionally, it has been hypothesized that anionic heparan sulfate proteoglycan components contribute to an electric barrier for macromolecules (Kasinath and Kanwar, 1993). The glomerular filter is affected in a large number of acquired and inherited diseases resulting in extensive leakage of plasma albumin and larger proteins leading to nephrotic syndrome and end stage renal disease. Understanding of the molecular mechanisms of the glomerular filtration process and its pathology is of fundamental importance for clinical medicine, which, in turn, may facilitate novel developments for diagnosis and treatment of complications in primary and secondary diseases of the kidney. Genetic diseases with defects in the filtration barrier as major symptoms can serve as models for providing such knowledge.

Congenital nephrotic syndromes (NPHS) form a heterogenous group of diseases characterized by massive proteinuria at or shortly after birth (Rapola et al., 1992). Nephrotic syndrome can be primary, acquired, or a part of other syndromes. Congenital nephrotic syndrome of the Finnish type (CNF, NPHS1) is a distinct entity among NPHS. It is an autosomal recessive disorder with an incidence of 1:10,000 births in Finland, but considerably less in other countries (Norio, 1966; Huttunen, 1976). The disease manifests itself already at the fetal stage with heavy proteinuria in utero, demonstrating early lesions of the glomerular filtration barrier. The pathogenesis of NPHS1 has remained obscure. There are no pathognomonic pathologic features, the most typical histological finding of NPHS1 kidneys being dilation of the proximal tubuli (Huttunen et al. 1980). The kidneys are also large and have been found to contain a higher amount of nephrons than age-matched controls (Tryggvason and Kouvalainen, 1975). Electron microscopy reveals no abnormal features of the GBM itself, although there is a loss of foot processes of the glomerular epithelial cells, a finding characteristic for nephrotic syndromes of any cause. Analyses of GBM proteins, such as type IV collagen, laminin, and heparan sulfate proteoglycan have not revealed abnormal findings in NPHS1 (e.g. see Ljungberg et al. 1993, Kestilä et al. 1994a). NPHS1 is a progressive disease, usually leading to death during the first two years of life, the only life-saving treatment being kidney transplantation (Holmberg et al. 1995). Importantly, most transplanted patients have, thus far, not developed extrarenal complications, suggesting that the mutated gene product is highly specific for kidney development and/or glomerular filtration function. However, about 20% of the patients have developed post-transplantation nephrosis the cause of which is unknown (Laine et al., 1993; Holmberg et al., 1995).

Due to its high specificity for the glomerular filtration process, NPHS1 provides a unique model disease for studies on this important kidney function. Since there was no strong candidate gene for the disease, we have used the positional cloning approach in our attempts to identify the CNF gene, and have localised the gene to a 150 kb region on chromosome 19q13.1 (Kestilä et al., 1994b; Männikkö et al., 1995). We have identified a novel gene in the critical region and shown it to be mutated in NPHS1. The gene product is a novel transmembrane protein, which in the human embryo shows a high expression level in renal glomeruli.

SUMMARY OF THE INVENTION

The present invention provides for the novel protein Nephrin and the gene encoding for this protein. The present invention encompasses a novel DNA nucleic acid sequence which is the nucleic acid sequence of SEQ ID NO: 1 which encodes for the nephrin protein. The present invention also encompasses the protein encoded for by the coding regions of the nucleic acid sequence of SEQ ID NO: 1 which has the amino acid sequence of SEQ ID NO: 2. In particular, the present invention also encompasses the mature nephrin protein in which the signal peptide has been cleaved off.

The present invention encompasses method, reagents and kits for screening individuals for the presence of mutated Nephrin gene for diagnosis, pre-natal screening, or post-natal screening for susceptibility to glomerular nephrosis or basement membrane disease. In particular, the present invention provides for screening for congenital nephrotic syndromes of the Finnish type (NPHS1).

The present invention provides for methods, reagents and kits for the therapeutic treatment of basement membrane disease associated with defective endogenous Nephrin gene product. Thus the present invention provides for therapeutic treatment using Nephrin protein, and in particular using protein produced by recombinant DNA methods. In addition, the present invention provides for gene therapy using therapeutic nucleic acid constructs containing the Nephrin gene, or substantially similar DNA sequence thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood in view of the attached drawings wherein:

FIG. 1 is a drawing showing a physical map of the NPHS1 locus at 19q13.1 and genomic organization of the NPHS1 gene. FIG. 1A, is a physical map of the 920 kb region between markers D19S208 and D19S224. FIG. 1B, is a diagram of overlapping cosmid clones spanning the 150 kb critical region containing the NPHS1 gene. Location of polymorphic markers are indicated by arrows. FIG. 1C, is a diagram showing the location of five genes, NPHS1, APLP1, A, B, C, characterised and searched for mutations in this study. FIG. 1D, is a drawing showing a schematic structure of the NPHS1 gene.

FIG. 2 shows a northern blot analysis of nephrin expression (the NPHS1 gene product) with mRNA from human embryonic and adult tissues. The northern filters containing 2 ug of human poly(A) RNA from four fetal and eight adult tissues (Clontech) were hybridized with a 1,371 bp nephrin cDNA probe (exons 1-10) made by RT-PCR from fetal kidney poly(A) RNA. FIG. 2A, shows distinct expression can be seen only with fetal kidney RNA (arrow). FIG. 2B, shows results using RNA from adult tissues, intense signal is only observed in a 4.3 kb band with kidney RNA (arrow), the other tissues exhibiting only insignificant if any positive signals. The tissues studied are marked above the filter and molecular size markers (kb) are shown to the sides of the filters.

FIG. 3 is a diagram of mutation analysis of the NPHS1 gene. Left: (A) Pedigree of an NPHS1 family with an affected child having a 2-bp deletion in exon 2. Sequences of the deletion point shown from patient (homozygous), parent (heterozygous) and a healthy sibling. Right: (B) Pedigree of an NPHS1 family with an affected child having a nonsense mutation in exon 26. Sequences of the mutated region are shown from patient (homozygous), parent (heterozygous) and a healthy sibling.

FIG. 4 is a diagram of the nucleotide-derived amino acid sequence of nephrin (the NPHS1 gene product) and predicted domain structure. FIG. 4A (SEQ ID NO: 2), is the predicted N-terminal signal sequence is 22 residues, the cleavage site being marked with an arrow. A putative transmembrane domain (spanning residues 1,059-1086) is shown in bold and underlined. The putative extracellular part of the protein contains eight Ig-like modules (boxed), and one fibronectin type III-like module adjacent to the transmembrane domain (boxed with a bold line, residues 941-1025). Cysteine residues are indicated by black dots and the ten putative N-glycosylation sites in the extracellular part of the protein are underlined. FIG. 4B shows the predicted domain structure of normal nephrin and the predicted effects of the two mutations (Fin-major and Fin-minor) identified in this study. The Ig-like modules are depicted by partial circles and the fibronectin type III like-motif by a hexagon. The transmembrane domain is shown as a black rectangle located in a membrane lipid bilayer. The locations of two free cysteine residues are indicated by lines with a black dot at the end. The Fin-major mutation would result in the production of part of the signal peptide and a short nonsense sequence. The Fin-minor mutation would result in a nephrin molecule lacking a part of the cytosolic domain.

FIG. 5 shows the results of expression of nephrin mRNA in human embryonic kidney by in situ hybridization. FIG. 5A, shows intense expression in glomeruli throughout the renal cortex, little if any specific expression being observed in other structures. (4× objective magnification). FIG. 5B, is a view at higher magnification which reveals intense expression in the periphery of individual glomeruli (straight arrows), probably mainly in epithelial cells. No expression is observed in the Bowman's capsule (bent arrow), proximal tubuli (open arrows), or endothelial cells of vessel walls. (20× objective magnification).

DETAILED DESCRIPTION OF THE INVENTION

Congenital nephrotic syndrome of the Finnish type (CNF, NPHS1, MIM 256300) is an autosomal recessive disorder, and a distinct entity among congenital nephrotic syndromes. It is characterized by massive proteinuria at the fetal stage and nephrosis at birth. Importantly, NPHS1 appears to solely affect the kidney and, therefore, it provides a unique model for studies on the glomerular filtration barrier. The NPHS1 gene has been localized to 19q13.1, and in the present study linkage disequilibrium was used to narrow the critical region to 150 kilobases which were sequenced. At least 10 novel genes, and one encoding amyloid precursor like protein were identified in this region. Five of the genes, all of which showed some expression in kidney, were analyzed by sequencing all their 63 exons in NPHS1 patients. Two mutations, a 2-bp deletion in exon 2 and a single base change in exon 26, both leading to premature stop codons were found in a novel 29-exon gene. The mutations were found either as homozygous or compound heterozygous in 44 out of 49 patients, 4 patients having the 2 bp deletion in one allele, the other potential mutation still being unknown. None among controls was found homozygous or compound heterozygous for the mutations. The gene product, termed nephrin, is a 1,241-residue putative transmembrane protein of the immunoglobulin family of cell adhesion molecules which by northern and in situ hybridization was shown to be kidney glomerulus-specific. The results demonstrate a crucial role for nephrin in the development or function of the kidney filtration barrier.

The invention will be more clearly understood by examination of the following examples, which are meant by way of illustration and not limitation.

Example 1 Methods and Procedures

Sequencing of Cosmid Clones

Isolation of cosmid clones spanning the region between D19S208 and D19S608 has been reported previously (Olsen et al., 1996). DNA of cosmid clones F19541, R33502, F15549, R28051, F19399, R31158 and R31874 was mechanically sheared by nebulization and fragments of 1000-2000 bp were isolated and subcloned into M13 phage, prior to random sequencing using ABI 377 automated DNA sequencers.

Analysis of Sequence

In order to develop new microsatellite markers, repeat regions were searched from the sequence, and three of them (D19S1173, D19S1175, D19S1176) were found to be polymorphic. Homology comparisons were performed using BLASTX and BLASTN programs (Altschul et al., 1990). Prior to BLASTN analyses, the nucleotide sequence was filtered using CENSOR (Jurka et al., 1996) to mask out repeat regions like Alu sequences. Exon prediction was made using GRAIL II (Uberbacher and Mural, 1991), GENSCAN (Burge and Karlin, 1997), FGENEH and HEXON (Solovyeh et al., 1994) programs, and prediction of the protein structure was made using BLASTP (Altschul et al., 1990) and EXPASY molecular biology server (Appel et al., 1994). The mutation search was performed by comparing patient sequences to the normal genomic sequence using the FASTA program of the GCG package (Genetics Computer Group, 1996).

Isolation of cDNAs

cDNAs were generated by PCR from poly(A) RNA from different tissues using primers based on the exon sequences. The PCR fragments were sequenced and used for screening of cDNA libraries. Marathon ready cDNA kits (Clontech Laboratories) were also used to characterize the 5′ and 3′ extremities of the cDNAs. Comparison of the cDNA and genomic sequences were made to establish the sizes of introns, as were intron sequences at acceptor and donor splice sites.

Southern and Northern Blots and in Situ Hybridization Analyses

For Southern analyses samples containing 10 μg of genomic DNA were digested with different restriction enzymes and electrophoreses on 1% agarose gels, transferred to nylon membranes and hybridized with the cDNA probe. In multiple-tissue northern analysis poly(A) RNAs from 8 adult and 4 fetal tissues were studied (Clontech). Hybridization was done in ExpressHyb buffer at 65° C. using a cDNA clone containing exons 1-10.

For in situ hybridization a fragment from the NPHS1 cDNA clone (corresponding to exon 10) was labeled with digoxigenin (Boehringer Mannheim), cut to about 150 base pair fragments by alkaline hydrolysis, and then used as a probe. Tissue sections of 7 μm from a 23-week human embryonic kidney were treated with 0.2M HCl, 0.1M triethanolamine buffer, pH 8.0, containing 0.25% (v/v) acetic anhydride and 100 μg/ml proteinase K. The sections were hybridized with the probe at 62° C. for 16 h. After rinsing in 50% formamide and standard sodium citrate, the probe was immunologically detected with an antibody to digoxigenin conjugated to alkaline phosphate enzyme (Boehringer Mannheim). The color was developed with NBT and BCIP.

Mutation Analysis

In this study we analyzed 49 Finnish NPHS1 patients, their parents and a total of 54 healthy siblings. The diagnosis of NPHS1 is based on severe proteinuria, a large placenta (>25% of birth weight), nephrotic syndrome during the first weeks of life, and exclusion of other types of congenital nephrotic syndrome (Koskimies 1990). Additionally, samples from 83 control individuals were analysed.

The NPHS1 gene was analysed by PCR-amplifying and sequencing all exon regions from genomic DNA. The sequences of the primers for exon 2 were 5′GAGAAAGCCAGACAGACGCAG3′ (5′ UTR)(SEQ ID NO: 3) and 5′AGCTTCCGCTGGTGGCT3′ (intron 2) (SEQ ID NO: 4), and the sequences of the primers for exon 26 were 5′CTCGGGGAGACCCACCC3′ (intron 23) (SEQ ID NO: 5) and 5′CCTGATGCTAACGGCAGGGC3′ (intron 26) (SEQ ID NO: 6). PCR reactions were performed in a total volume of 25 ul, containing 20 ng of template DNA, 1× AmpliTaq buffer (Perkin-Elmer), 0.2 mM of each nucleotide, 50 ng of primers and 0.5 U AmpliTaq Gold DNA polymerase. The reactions were carried out for 30 cycles with denaturation at 95° C. for 1 min, annealing at 60° C. for 1 min, and extension at 72° C. for 1 min. In the first cycle denaturation was carried out for 12 min, and extension in the last cycle was for 8 min. PCR products were separated by 1.5% agarose gel, sliced off and purified by the QiaexII system (Qiagen). The purified PCR product was sequenced using specific primers employing dRhodamine dye-terminator chemistry and an ABI377 automated sequencer (Perkin-Elmer).

When screening for the NPHS1 Fin-major mutation from parents, siblings and controls, a 100 bp PCR product containing the exon 2 deletion site was amplified using a radioactively end-labeled primer, and electrophoresed on 6% polyacrylamide gels. The second NPHS1 Fin-minor mutation could be screened for using a novel restriction site for DdeI. The 140 bp amplified PCR product was digested with DdeI and the products (140 bp or 90 bp+50 bp) were separated on an agarose gel (1% SeaKem agarose-3% NuSieve agarose).

In general, methods and procedures for performing molecular biological and biochemical techniques are known in the art and can be found in available texts and references, such as for example Sambrook et al., (1989) Molecular Cloning: a laboratory manual, 2nd edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Short Protocols in Molecular Biology, 2nd edition (edited by Ausubel et al., John Wiley & Sons, New York, 1992); Davis et al., (1986) Basic Methods in Molecular Biology (Elsevier, N.Y.); Gene Expression Technology (edited by David Goeddel, Academic Press, San Diego, Calif., 1991).

Example 2 Characterization of Genes at the CNF Locus

Following localisation of the NPHS1 gene to 19q13.1, overlapping cosmid clones from the interval of interest between markers D19S208 and D19S224 were isolated (Männikkö et al. 1995; Olsen et al., 1996). Based on the significant linkage disequilibrium observed with D19S608 and D19S610, as well as the new microsatellite markers, D19S1173, D19S1175, and D19S1176, identified in this study, the NPHS1 gene was fine-mapped between D19S1175 and D19S608, in close vicinity of D19S1176 and D19S610 (FIG. 1). Southern hybridization analyses of NPHS1 patient DNA with genomic clones did not reveal variations, suggesting that the NPHS1 mutations do not represent major genomic rearrangements. The 150 kb critical region was sequenced in its entirety, and the sequence was searched for potential candidate genes using exon prediction programs and data base similarity searches. Based on those analyses, the critical region was estimated to include over 100 potential exons. Similarity searches revealed one previously known gene, i.e. APLP1 encoding an amyloid precursor-like protein (Lenkkeri et al., in press) and eight distinct expressed sequence tags (ESTs). Together the analyses indicated the presence of at least ten novel genes in the critical region.

FIG. 1 illustrates a physical map of the NPHS1 locus at 19q13.1 and genomic organisation of the NPHS1 gene. FIG. 1A, Physical map of the 920 kb region between D19S208 and D19S224. FIG. 1B, Overlapping cosmid clones spanning the 150 kb critical region containing the NPHS1 gene. Location of polymorphic markers are indicated by arrows. FIG. 1C, Location of five genes, NPHS1, APLP1, A, B, C, characterised and searched for mutations in this study. FIG. 1D, Schematic structure of the NPHS1 gene.

Using Grail and Genscan exon prediction programs and sequences from cDNAs, the exon/intron structures of five of the genes, NPHS1 (FIG. 1), APLP1, A, B, and C (not shown) were determined. Although steady state transcript levels varied, northern analyses revealed expression of all the genes in kidney, and with the exception of NPHS1, also in other tissues. Therefore, none of them could be excluded as the NPHS1 gene and all were subjected to mutation analysis.

Example 3 Identification of the NPHS1 Gene

Haplotype analyses of NPHS1 chromosomes have revealed two major classes in Finnish patients (Männikkö et al., 1995; this study). The first one containing haplotypes 1-1-1-6-g-2-8-9 and 1-1-1-6-g-6-4-2 (markers D19S1173, D19S1175, D19S1176, D19S610, RFLP of gene B, D19S608, D19S224, D19S220, respectively) is the most common one found in 78% of Finnish NPHS1 chromosomes. The second haplotype class, 3-5-3-6-a-8-10-x, is found in 13% of cases. The remaining 9% of observed haplotypes show totally different allele combinations, and have been thought to represent other mutations. Two major haplotype classes could represent the same mutation, because they both share allele 6 of D19S610. However, the present results demonstrated that they represent two different mutations.

Since Southern hybridization analyses did not reveal any major gene rearrangements, mutations were searched by direct sequencing of PCR-amplified exon regions of, if necessary, all the genes of this region.

The 17 exon APLP1 gene located distal to D19S610 did not show variations between patients and controls, and was excluded as the NPHS1 gene (Lenkkeri et al., in press). Also, the novel genes A, B and C, containing 9, 5 and 3 exons, respectively, did not have sequence variants segregating with NPHS1, and could similarly be excluded as the NPHS1 genes (data not shown). A fourth novel gene (NPHS1) located proximal to D19S610 encoding a transcript of about 4.3 kb was shown to be strongly expressed in human embryonic and adult kidneys, no clear signals above background being observed in other tissues (FIG. 2).

FIG. 2 illustrates the results of Northern analysis of nephrin expression with mRNA from human embryonic and adult tissues. The northern filters containing 2 ug of human poly(A) RNA from four fetal and eight adult tissues (Clontech) were hybridized with a 1,371 bp nephrin cDNA probe (exons 1-10) made by RT-PCR from fetal kidney poly(A) RNA. In FIG. 2A, Distinct expression can be seen only with fetal kidney RNA (arrow). In FIG. 2B, Using RNA from adult tissues, intense signal is only observed in a 4.3 kb band with kidney RNA (arrow), the other tissues exhibiting only insignificant if any positive signals. The tissues studied are marked above the filter and molecular size markers (kb) are shown to the sides of the filters.

Therefore, this gene was a strong candidate for NPHS1. Full-length cDNA for the transcript was constructed using fetal kidney poly(A) mRNA (Clontech) and PCR primers made based on the predicted exon structure. The gene was found to have a size of 26 kb and to contain 29 exons (FIG. 1).

Exon sequencing analyses revealed the presence of two major mutations in over 90% of NPHS1 chromosomes (FIG. 3). FIG. 3 illustrates mutation analysis of the NPHS1 gene. Left: (A) Pedigree of a NPHS1 family with an affected child having a 2-bp deletion in exon 2. Sequences of the deletion point shown from patient (homozygous), parent (heterozygous) and a healthy sibling. Right: (B) Pedigree of a NPHS1 family with an affected child having a nonsense mutation in exon 26. Sequences of the mutated region are shown from patient (homozygous), parent (heterozygous) and a healthy sibling.

The first mutation, a 2-bp deletion in exon 2 causes a frameshift resulting in the generation of a stop codon within the same exon. This mutation was found in all NPHS1 chromosomes with the haplotype 1-1-1-6-g-2-8-9 and 1-1-1-6-g-6-4-2 (total of 76 chromosomes). One out of 83 control individuals was heterozygous for the Fin-major mutation. The second sequence variant found in the NPHS1 gene was a nonsense mutation CGA.fwdarw.TGA in exon 26, present in patients with haplotype 3-5-3-6-a-8-10-x (13 chromosomes), and three patients with different haplotypes. None of the parents, healthy siblings, or controls (total of 230 individuals) were homozygous or compound heterozygous for the two mutations identified here. Since the gene cloned in this study is the one involved in a hereditary nephrotic syndrome, we refer to it as NPHS1 gene.

Out of 49 NPHS1 patients studied, 32 were homozygous for the 2-bp deletion in exon 2 (Fin-major), four were homozygous for the nonsense mutation in exon 26 (Fin-minor), and eight were compound heterozygotes. Four patients had the Fin-major mutation in one allele, the other potential mutation still being unknown. One patient had neither one of the two mutations.

Example 4 Characterization of the NPHS1 Gene Product

The cDNA-predicted amino acid sequence of the NPHS1 protein (nephrin) is 1,241 residues (FIG. 4), with a calculated molecular mass of 134,742 without posttranslational modifications.

FIG. 4 shows nucleotide-derived amino acid sequence of nephrin and predicted domain structure (the NPHS1 gene product). FIG. 4A illustrates the predicted N-terminal signal sequence is 22 residues, the cleavage site being marked with an arrow. A putative transmembrane domain (residues 1,059-1086) is shown in bold and underlined. The putative extracellular part of the protein contains eight Ig-like modules (boxed), and one fibronectin type III-like module adjacent to the transmembrane domain (boxed with a bold line). Cysteine residues are indicated by black dots and the ten putative N-glycosylation sites in the extracellular part of the protein are underlined. FIG. 4B illustrates predicted domain structure of normal nephrin (the NPHS1 gene product) and the predicted effects of the two mutations (Fin-major and Fin-minor) identified in this study. The Ig-like modules are depicted by partial circles and the fibronectin type III like-motif by a hexagon. The transmembrane domain is shown as a black rectangle located in a membrane lipid bilayer. The locations of three free cysteine residues are indicated by lines with a black dot at the end. The major NPHS1 mutation would result in the production of a secreted protein containing only a part of the first Ig-like module. The Fin-minor mutation would result in a nephrin molecule lacking a part of the cytosolic domain.

Several similarity comparison and protein structure prediction programs predicted that the NPHS1 protein would be a transmembrane protein of the immunoglobulin superfamily. There is a tentative 22-residue-long N-terminal signal peptide, an extracellular domain containing eight immunoglobulin-like domains, one fibronectin type III domain-like module, followed by a single putative transmembrane domain-like sequence, and a cytosolic C-terminal end. In spite of the presence of known structural modules (FIG. 4), the sequence identity with corresponding domains of proteins in the data base was relatively low. The tentative extracellular portion of the protein contains ten NXS or NXT consensus triplets for N-glycosylation. Furthermore, there are seven SG doublets, that are potential attachment sites for heparan sulfate.

Northern hybridization analysis carried out with poly(A) mRNA from four human embryonic and eight adult tissues revealed a high steady state level of the NPHS1 gene transcript in the kidney, but not notably in other tissues. (FIG. 2). In situ hybridization carried out on a kidney sample from a 23-week-old human embryo revealed intense expression signals in the glomeruli (FIG. 5A). At higher magnification (FIG. 5B), the signals could be seen in the periphery of mature and developing glomeruli, while the central mesangial regions are negative. It is apparent that the positive cells are epithelial podocytes. No specific signals were obtained with the antisense control probe.

FIG. 5 illustrates expression of nephrin mRNA in human embryonic kidney by in situ hybridization FIG. 5A shows intense expression is seen in glomeruli throughout the renal cortex, little if any specific expression being observed in other structures. (4× objective magnification). FIG. 5B, Higher magnification reveals intense expression in the periphery of individual glomeruli (straight arrows), probably mainly in epithelial cells. No expression is observed in the Bowman's capsule (bent arrow), proximal tubuli (open arrows), or endothelial cells of vessel walls. (20× objective magnification).

Example 5 The NPHS1 Gene and its Gene Product Nephrin

Several lines of evidence obtained in the present study show that we have positionally cloned the gene affected in congenital nephrotic syndrome of the Finnish type. First, the defective gene is located in the critical 150 kb region on chromosome 19q13.1 to which the gene has been localized using linkage disequilibrium analyses (Kestilä et al., 1994b; Männikkö et al., 1995; Kestilä et al. manuscript). Second, the two mutations identified in the study were shown to be present, either as homozygous or compound heterozygous mutations, in 44 out of 49 Finnish patients studied. Four of the remaining patients had the major mutation in one allele, the mutation in the other allele being, as yet, unidentified. One patient who did not have either of the two mutations, has a unique haplotype and, therefore, probably carries a different mutation. Third, individuals homozygous or compound heterozygous for the mutations were not found in 230 control DNAs. Additional, indirect evidence was the strong and practically renal glomeruli-specific expression of the gene, which implies involvement of the gene product in glomerular development or function.

Identification of the NPHS1 Gene

The present identification of the NPHS1 gene demonstrates the power of linkage disequilibrium analysis and direct DNA sequencing in the positional cloning of disease genes containing small mutations. Here, linkage disequilibrium mapping (Hstbacka et al., 1994) which when used with DNA from individuals of a homogenous population, such as the isolated Finnish population (de la Chapelle, 1993), was utilized to localize the NPHS1 gene to a 150 kb genomic segment. In order to find genes located in this region, the entire segment was first sequenced, and using a combination of exon prediction programs and homology comparison analyses we could construct remarkably accurate gene structures that were verified from cDNAs. These cDNAs could be isolated either with the use of EST clones or by using the predicted exon sequences to construct cDNAs by PCR from mRNA. In this manner we could quickly identify 11 genes within the 150 kb NPHS1 containing genomic segment. Since none of the genes was an obvious candidate for NPHS1, and no major gene rearrangements, such as deletions, insertions or inversions, were found in patient DNAs, search for small mutations had to be initiated, if necessary, in all the 11 genes. Having determined the exon and cDNA sequences for the genes, methods such as SSCP and DGGE, which are frequently used for identification of small mutations, were potential alternatives. However, our experience from the search for small mutations in Alport syndrome (Barker et al., 1990; Tryggvason, 1996) suggests that these methods can frequently yield false negatives. For example, SSCP analyses in quite large patient populations have revealed only a 35-50% mutation detection rate (Kawai et al., 1996, Knebelmann et al. 1996, Renieri et al., 1996), while our direct sequencing of PCR-amplified exon regions has yielded over 80% detection. We therefore decided to use direct sequencing of exon regions to find the NPHS1 mutations. Although we had to sequence numerous exons of several genes, this relatively soon resulted in the identification of two small mutations in one gene. We conclude that sequencing of even a large candidate gene region and direct sequencing of its genes is an attractive and, above all, reliable method to search for small mutations in positional cloning, particularly if only few mutations can be expected to be present.

Genetics of NPHS1

Crucial components in the successful positional cloning of the NPHS1 gene were the small isolated population, good clinical records and equal, high quality health care system which made it possible to reliably collect family samples. A typical situation in population isolates is that close to 100% of cases are caused by the same mutation, and this phenomenon can already be seen in haplotype analysis. Observed changes in the founder haplotype, caused by historical recombinations, can be used to restrict the critical chromosomal region to a short genomic segment. Thus, differences in the major NPHS1 haplotype 1-1-1-6-g-2-8-9 enabled substantial narrowing of the interval, leading to the isolation of the NPHS1 gene. The major NPHS1 mutation causes only 78% of cases, in contrast to many other “Finnish diseases” with 95-98% prevalence of major disease alleles (e.g. Ikonen et al., 1991). However, the two main NPHS1 mutations characterized in this study together represent 94% of Finnish cases.

Congenital nephrotic syndrome of the Finnish type is enriched in the Finnish population, but several cases can be found worldwide. Considerable immigration from Finland to Minnesota has also caused the spread of NPHS1 to the USA (Norio 1966; Mahan et al., 1984). In addition, several CNF cases have been diagnosed in different European countries, and linkage studies have supported association of analyzed families to the same chromosome 19 locus (Fuchshuber et al., 1996).

The identification of the NPHS1 gene and disease causing mutations have immediate clinical significance, as they have enabled the development of exact DNA-based diagnosis for NPHS1 and carrier screening. This is particularly important, as we have recently demonstrated that the screening method widely used in Finland for NPHS1 based on measurements of alpha-fetoprotein levels in amniotic fluid can lead to false positive results and subsequent abortions of healthy NPHS1 carriers (Männikkö et al., 1997).

Nephrin—a Glomerulus-specific Cell Adhesion Receptor

Due to the high association of expression and pathology with glomeruli, the proximal part of the nephron, we have named the NPHS1 gene product nephrin. The role of nephrin remains unknown, but it is likely to be an adhesion receptor and a signaling protein, as its domain structure resembles that of a large group of cell adhesion receptors belonging to the immunoglobulin superfamily (Brummendott and Rathjen, 1994).

The Ig-like domains of nephrin are all of type C2 which is particularly found in proteins participating in cell-cell or cell-matrix interactions. Between the sixth and seventh Ig-like domains there is a spacer of about 130 residues containing an unpaired cysteine, and there is another unpaired cysteine in the fibronectin type III-like domain. Their SH groups could be involved in the formation of cis homo/heterodimers, participate in thioether or thioester bonds with unknown structures, or be buried within the domains, as suggested by Brummendott and Rathjen (1994).

Data base searches revealed that the cytosolic domain that contains nine tyrosine residues of nephrin has no significant homology with other known proteins. However, sequence motifs surrounding tyrosines suggest that tyrosines 1176, 1192 and 1217 could become phosphorylated during ligand binding of nephrin (see, Songyang et al. 1993). In that case, binding sites for the SH2-domains of Src-family kinases, Abl-kinase, and an adaptor protein Nck might be created (tyrosines 1176 and 1192 are followed by the motif DEV, and tyrosine 1217 by DQV). The crucial role for the intracellular domain of nephrin is emphasized by the fact that the Fin-minor mutation which results in the loss of 132 out of 155 residues results in full blown NPHS1.

The pathogenesis of NPHS1 has been thought to primarily or secondarily involve the highly anionic glycosaminoglycans, as the content of such molecules that are considered important for the glomerular filtration process is reported to be decreased in the GBM in proteinuria (Kasinath and Kanwar, 1993). It cannot be excluded that nephrin is a proteoglycan, as it has several SG consensus sites for heparan sultate side chains, including the triplet SGD which is the major attachment sequence for the three large heparan sulfate side chains in the basement membrane proteoglycan perlecan (Noonan et al., 1991; Kallunki and Tryggvason, 1992; Dolan et al., 1997). However, thus far no Ig-like receptors have been reported to contain glycosaminoglycans.

How does nephrin function and what is its role in glomerular function? A vast majority of similar receptors interact with other membrane proteins in a homo- or heterophilic manner. However, some of the receptors have been shown to interact with extracellular matrix (ECM) proteins. For example, the myelin-associated glycoprotein MAG whose extracellular domain contains five Ig-like domains, interacts with different types of collagens and glycosaminoglycans (Fahrig et al., 1987). Furthermore, the axonal glycoprotein F11 and the deleted in colorectal cancer (DCC) protein have both been shown to bind tenascins and netrins, respectively (Zisch et al., 1992; Pesheva et al., 1993; Keino-Masu, 1996). Since it is possible that nephrin either binds another membrane protein or a protein of the ECM, which in this case would be the GBM, it will be important to localize nephrin by immunoelectron microscopy before embarking on the search for a specific ligand.

Whatever its function, the in situ hybridization analyses strongly suggested that nephrin is produced in glomerular epithelial cells that form the foot processes partially covering the outside of the glomerular capillaries. The ultimate filtration barrier for plasma macromolecules is located in the diaphragm covering the slit pores between the foot processes. In NPHS1 and nephrotic syndromes of other causes, fusion of the foot processes is a general finding, and the structure or function of the slit pores are somehow affected with proteinuria as a result. It is proposed that the plasma membrane protein nephrin is important for maintaining the integrity of the foot processes of glomerular epithelial cells, or is crucial for their anchorage to components of the GBM.

Conclusions

The identification of the NPHS1 gene will immediately find applications for diagnosis of the disease. Studies on the gene product nephrin, a putative cell adhesion and signaling receptor, may also provide a key to new fundamental knowledge on the molecular mechanisms of glomerular filtration, which despite decades of research are still poorly understood. As abnormal function of the filtration barrier is a major complication in many clinically important kidney diseases, such as diabetic nephropathy, nephrotic syndromes and glomerulonephritides, the present work is likely to have a more general impact on clinical nephrology. Immediate questions relate to the developmental expression and location of the protein, which would require the generation of antibodies and nucleotide probes for studies in animal and cell culture systems.

Example 6 Genetic Screening for Basement Membrane Disease

With the identification and characterisation of nephrin as a critical component in basement membrane disease associated with glomerular nephropathy, it is now possible to screen individuals, both pre- and post-natal screening, for susceptibility for basement membrane disease by detecting mutated nephrin gene or protein. Such information will be useful to medical practitioners for the future diagnosis of disease conditions in screened individuals, and for planning preventative measures for the possible containment of future disease. Such information will be useful for the diagnosis of currently active disease conditions. The present invention allows for the diagnosis of currently active disease conditions, as being related to basement membrane disease by detecting mutated nephrin gene or protein. The discovery of the nephrin gene provides a means for detecting the presence of the nephrin gene in individuals, and for the determination of the presence of any mutations in said gene. Such means for detection comprises nucleic acids having the entire nephrin gene sequence, or fragments thereof which will specifically hybridize to said nephrin gene, or mRNA transcripts from said nephrin gene under stringent conditions. An additional means for detection of the nephrin gene and mutations therein comprise specific contiguous fragments of said gene, and complementary gene sequence, which can be combined for use as primers for amplifying the targeted gene sequence. Said means for detection of mutations in a nephrin gene also comprise direct hybridization of normal gene with target gene and subsequent detection of successful hybridization. In all cases, the target gene may be amplified or unamplified DNA or RNA isolated from the individual to be tested.

Antibody Screening of Tissues and Samples

By having the NPHS1 gene sequence, it is well within the skill of one in the art to use existing molecular biology and biochemical techniques to construct and use an expression vector which will produce recombinant nephrin protein, or fusion protein, purify this protein, and produce antibodies specifically reactive with nephrin. The expression of proteins in bacterial, yeast, insect and mammalian cells is known in the art. It is known in the art how to construct and use expression vectors in which the expressed gene contains one or more introns. The production of monoclonal antibodies is well known in the art, and the use of polyclonal and monoclonal antibodies for immunohistochemical detection of protein in tissue samples is a routine practice. A wide variety of detectable labels are available for use in immunohistochemical staining and immunoassays for detection of protein in samples such as homogenised tissue, blood, serum, urine or other bodily fluids.

One of ordinary skill in the art will be able to readily use the teachings of the present invention to design suitable assays and detection schemes for practising the screening methods contemplated by the present invention.

Gene Therapy

Given the teaching of the present invention, it will be possible to address deficiencies in Nephrin gene or protein by gene therapy or therapy using recombinant protein. Methods for the administration of protein and gene therapy are known in the art.

GenBank Accession Numbers

The accession numbers for the cosmid clones characterised are:

-   F19541=U95090, R33502=AC002133, R28051=AD000864, F19399=AD000833,     R31158=AD000827, R31874=AD000823. The accession for the nephrin cDNA     sequence is AF035835.

One of ordinary skill in the art will be able to readily use the teachings of the present invention to design and construct suitable nucleic acid sequences which will be the functional equivalents of those disclosed. One of ordinary skill in the art will know that there exists many allelic variants of the disclosed nucleic acid sequences which still encode for a nephrin protein with equivalent function. The teaching of the present invention allows for the discovery of mutations in the nephrin gene and the modified protein therein encoded.

Example 7 Screening for Small Molecule Therapeutics

With the identification and characterisation of nephrin as a critical component in kidney pathology and proteinuria, and thus implicated in many kidney diseases, it is now possible to screen for small molecule therapeutics using nephrin and the nephrin gene. Screening for such therapeutics can be accomplished by sequential selective screening for activity and molecules which specifically hybridize to nephrin, or which specifically effect the expression of the nephrin gene. Selective screening can be performed on pools of small molecule compounds generated by standard combinatorial chemistry, on known molecules, or in combination with computer modeling of the nephrin protein structure and rational drug design. Such methods and techniques are known in the art.

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1. A method for detecting the presence or absence of a mutation in the coding region of the gene for congenital nephrotic syndrome of the Finnish type (NPHS1), comprising the steps of: a) analyzing all, or a portion of, the nucleotide sequence of the coding region of a NPHS1 gene contained in the nucleic acid test sample; b) comparing the results of the analysis of the nucleotide sequence of the NPHS1 gene coding region in the nucleic acid test sample of step a) with the results of an analysis of a control sample, wherein the control sample comprises a NPHS1 gene, and wherein the nucleotide sequence of the coding region of the control NPHS1 gene consists of SEQ ID NO: 1; and c) determining the presence or absence of differences between the nucleic acid sequence of the coding region of the NPHS1 gene in the nucleic acid test sample and SEQ ID NO: 1, wherein the determination of a difference between said nucleic acid sequence of the coding region of the NPHS1 gene in the nucleic acid test sample and SEQ ID NO: 1 is indicative of a mutation in the coding region of the test sample NPHS1 gene.
 2. The method of claim 1, wherein the coding region of the NPHS1 gene encodes a NPHS1 protein having the amino acid sequence of SEQ ID NO:
 2. 3. The method of claim 1, wherein the NPHS1 gene is amplified prior to analysis.
 4. A method for detecting the presence or absence of a mutation in the coding region of the gene for congenital nephrotic syndrome of the Finnish type (NPHS1), comprising the steps of: a) analyzing all, or a portion of, the nucleotide sequence of the coding region of a NPHS1 gene contained in a nucleic acid test sample; b) comparing the results of the analysis of the nucleotide sequence of the NPHS1 gene coding region in the nucleic acid test sample of step a) with the results of an analysis of the nucleotide sequence of SEQ ID NO: 1; and c) determining the presence or absence of differences between the nucleic acid sequence of the coding region of the NPHS1 gene in the nucleic acid test sample and SEQ ID NO: 1, wherein the determination of a difference between said nucleic acid sequence of the coding region of the NPHS1 gene in the nucleic acid test sample and SEQ ID NO: 1 is indicative of a mutation in the coding region of the test sample NPHS1 gene.
 5. The method of claim 4, wherein the coding region of the test sample NPHS1 gene is amplified prior to analysis.
 6. The method of claim 1 wherein analyzing all, or a portion of, the nucleotide sequence of the coding region of the NPHS1 gene comprises sequencing all, or a portion of, the coding region of the NPHS1 gene in the test sample.
 7. The method of claim 1 wherein analyzing all, or a portion of, the nucleotide sequence of the coding region of the NPHS1 gene comprises hybridizing all, or a portion of, the coding region of the NPHS1 gene in the test sample with a nucleic acid probe comprising all, or a portion of, SEQ ID NO: 1 under conditions suitable for hybridization and detecting the presence or absence of a hybridization product, wherein the presence of a hybridization product is indicative of the absence of a mutation in the coding region of the NPHS1 gene, and the absence of a hybridization product is indicative of the presence of a mutation in the coding region of the NPHS1 gene.
 8. The method of claim 4 wherein analyzing all, or a portion of, the nucleotide sequence of the coding region of the NPHS1 gene comprises sequencing all, or a portion of, the coding region of the NPHS1 gene contained in the nucleic acid test sample.
 9. The method of claim 4 wherein analyzing all, or a portion of, the nucleotide sequence of the coding region of the NPHS1 gene in the test sample comprises hybridizing all, or a portion of, the coding region of said NPHS1 gene with a nucleic acid probe comprising all, or a portion of, SEQ ID NO: 1 under conditions suitable for hybridization and detecting the presence or absence of a hybridization product, wherein the presence of a hybridization product is indicative of the absence of a mutation in the coding region of the NPHS1 gene, and the absence of a hybridization product is indicative of the presence of a mutation in the coding region of the NPHS1 gene.
 10. The method of claim 3 wherein a portion of the amplified NPHS1 gene is analyzed for the presence of a DdeI restriction site by digesting the amplified gene products with DdeI and detecting the digestion products.
 11. The method of claim 5 wherein a portion of the amplified NPHS1 gene is analyzed for the presence of a DdeI restriction site by digesting the amplified gene products with DdeI and detecting the digestion products.
 12. The method of claim 3 wherein all, or a portion of, the amplified NPHS1 gene is analyzed by electrophoresis.
 13. The method of claim 5 wherein all, or a portion of, the amplified NPHS1 gene is analyzed by electrophoresis. 